INTRODUCTION

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Major histocompatibility complex class II (MHC II) molecules are crucial players in the immune response. These cell surface glycoproteins are constitutively expressed on antigen-presenting cells and can be induced on other cell types by gamma interferon (4, 5, 9, 20). They present processed antigens to helper T cells and initiate immune responses (10). Different subtypes of human MHC II molecules are transcribed from TATA-less promoters that contain conserved S, X, and Y boxes (4, 5, 9, 14, 16, 20). Protein complexes that bind to these proximal promoter elements finally attract the class II transactivator (CIITA) by an as-yet-unknown mechanism (4, 5, 9, 14, 16, 20, 33). S and X boxes bind a tripartite regulatory factor X (RFX) complex, while the Y box binds the nuclear factor Y (NFY) complex (14). Congenital absence of MHC II molecules on B cells is known as the bare lymphocyte syndrome (BLS) (20). Its unique phenotypic outcome is the result of diverse genetic backgrounds. While the genes for MHC II determinants remain intact, different mutations have been found in four trans-acting factors, namely, RFX5, RFXAP, RFXANK(B), and CIITA, defining four complementation groups of BLS (12, 22, 24, 31).

The complex architecture of proteins that are directly or indirectly bound to MHC II promoters is achieved by multiple protein-protein interactions within the RFX and NFY complexes, between these complexes, and with CIITA, the master switch that triggers the transcription of MHC II genes (8, 11, 14, 33). The RFX complex is composed of three subunits, namely, RFX5, RFXAP, and RFXANK(B). We and others have shown how the RFX complex assembles (11, 26). Whereas RFXAP interacts with the two other subunits via its C-terminal, glutamine-rich domain (11, 26), RFX5 contacts the RFX complex via two separate regions that surround its DNA-binding domain (11). RFXANK or RFX(B) (henceforth called RFXANK) is a 33-kDa protein with three distinct domains (11, 22, 24). The potential role of its N-terminal imperfect PEST sequence is still unknown. The C-terminal portion of RFXANK contains at least three ankyrin repeats. RFXANK is therefore the only protein within the RFX complex that contains well-established modules for protein-protein interactions. The domain between the PEST-like sequence and the first ankyrin repeat has been suggested to make contacts with DNA, although it lacks any recognizable DNA-binding consensus sequence.

Ankyrin repeats are one of the most common protein sequence motifs, with each of them consisting of 33 residues (30). They have been found in proteins as different as Cdk inhibitors, signal transduction and transcriptional regulators, cytoskeletal organizers, developmental regulators, and toxins. Their presence in such a colorful palette of functionally diverse proteins suggests that their role is of more of a structural than a functional nature. Indeed, these protein scaffolding modules mediate protein-protein interactions in a number of different biological systems, from microbes to humans (30). The number of ankyrin repeats varies from only 2 in plutonium, a small protein from Drosophila (2), to more than 20 in ankyrin, a well-studied ubiquitous adapter protein that links membrane proteins with the spectrin-based cytoskeleton (23). Elucidation of the three-dimensional structure of the ankyrin repeats by X-ray crystallography and nuclear magnetic resonance techniques offered an insight into their conserved, stable backbone. Certain amino acid residues of the backbone play only an architectural role by making multiple intramolecular interactions, mainly hydrogen and hydrophobic ones. However, ankyrin repeats can easily handle a broad diversity of their binding partners by containing variable residues, insertions, and deletions between single repeats and by stacking in different numbers. Thus, it is not surprising that there are no specific ankyrin-binding motifs in their target proteins, which can also vary considerably in their shape and size (30). Potential binding surfaces on the ankyrin repeats are all solvent-exposed parts that contain variable residues.

Although it has been suggested that the ankyrin repeats of RFXANK mediate protein-protein interactions within the RFX complex (11, 26), no informative mapping on RFXANK has been done. Furthermore, the involvement of ankyrin repeats in protein-protein interactions that go beyond the RFX complex has not been addressed. Our preliminary experiments showed that RFXANK binds multiple protein partners. We wanted to investigate how the smallest subunit of the RFX complex successfully mediates these protein-protein interactions and what is the role of ankyrin repeats in this process. However, deletion mapping was not informative, and we found the structure-function analysis, based on a three-dimensional structure prediction for RFXANK, more useful. By combining a well-studied ankyrin fold with site-directed alanine mutagenesis, we showed how its multiple binding sites recruit the interacting proteins, and in this way we mapped precisely the ankyrin-centered interactions on MHC II promoters.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Cell culture. COS cells were grown in Dulbecco's modified Eagle's medium. Media were supplemented with 10% fetal bovine serum, 100 mM L-glutamine, and 50 µg each of penicillin and streptomycin per ml.

Plasmid constructions. Myc epitope-tagged pEF-RFXANK and hemagglutinin (HA) epitope-tagged pEF-RFXAP plasmid constructs were generated as described before (26). HA epitope-tagged wild-type CIITA protein was generated by PCR and inserted into the EcoRI-SpeI sites of the modified pEF-BOS vector (1). The glutathione S-transferase (GST)-RFXANK plasmid construct was described before (26). Deletion mutants of RFXANK were created by PCR. The primer sequences were as follows: the forward primer F (5'-GCTTCGGGATCCATGGAGCTTACCCAGCCTGCA-3') and the reverse primers R1 (5'-GCTTCGGAATTCCTACTGGAAGAGCTTGAGGATGTG-3') for RFXANK(1-251), R2 (5'-GCTTCGGAATTCCTAGCCTCGGGCCAGCAAGGCCTC-3') for RFXANK(1-213), R3 (5'-GCTTCGGAATTCCTAGTCACGCTCCAGCAGCAGCCC-3') for RFXANK(1-180), and R4 (5'-GCTTCGGAATTCCTAACCCCACTCCAGCAGGAAGCG-3') for RFXANK(1-147). Amplified products were ligated into the BamHI-EcoRI sites in frame with the coding region of the GST gene in pGEX-2TK (Amersham Pharmacia Biotech, Piscataway, N.J.). All cDNAs were confirmed by DNA sequencing. The pT7T3-RFXAP and pSV-CIITA plasmid constructs were described before (13, 26).

Site-directed mutagenesis. Mutagenesis of the ankyrin repeats of RFXANK was performed by using a Transformer Site-Directed Mutagenesis Kit (Clontech Laboratories, Palo Alto, Calif.) according to the manufacturer's instructions. The template for mutagenesis was the GST-RFXANK plasmid construct. The mutagenic primers were designed as follows: 5'-CCTCGTCAACAAGCCAGCGGCCGCGGCCTTCACCCCCCTC-3' for GST-RFXANK-1 (contains D121A, E122A, R123A, and G124A substitutions in the cDNA of the wild-type RFXANK protein), 5'-GCCGACCCCCACATCCTGGCGGCCGCGGCCGAGAGCGCCCTGTCG-3' for GST-RFXANK-2 (contains K155A, E156A, and R157A substitutions), 5'-GGACATCAACATCTATGCGGCCGCGGCCGGGACGCCACTGC-3' for GST-RFXANK-3 (contains D187A, W188A, N189A, and G190A substitutions), 5'-GCTGACCTCACCACCGAAGCCGCGGCCGCGTACACCCCGATGG-3' for GST-RFXANK-4 (contains D221A, S222A, and G223A substitutions), 5'-GAGAGATTGAGACCGTTGCGTTCCTGCTGGCGGCCGGTGCCGACCCCCAC-3' for GST-RFXANK-OH1 (contains R141A, E145A, and W146A substitutions), 5'-GTGGGGCTGCTGCTGGCGGCCGACGTGGACATCAACATCTATGATTGG-3' for GST-RFXANK-OH2 (contains G174A, E178A, and R179A substitutions), 5'-CACGTGAAATGCGTTGCGGCCTTGCTGGCCGCGGGCGCTGACCTCACCAC-3' for GST-RFXANK-OH3 (contains E207A and R212A substitutions), 5'-GGAGGGACGCCACTGGCGGCCGCTGCGGCCGGGAACCACGTGAAATGCG-3' for GST-RFXANK-IH3 (contains L195A, Y196A, V198A, and R199A substitutions), 5'-GCACAGGCGGCTACACAGCCATTGTGGGGCTGCTGCTGG-3' for GST-RFXANK-turn2 (contains a D171A substitution), 5'-GCGCGGGAACCACGTGGCGTGCGTTGAGGCCTTGCTGGCCCG-3' for GST-RFXANK-turn3 (contains a K204A substitution), 5'-GGCCCTGGGATACCGGGCGGTGCAACAGGTGATCGAGAACC-3' for GST-RFXANK-turn4 (contains a K237A substitution), and 5'-GGAATGGAGGGACGCCACTGCCGTACGCTGTGCGCGGGAACCACG-3' for GST-RFXANK-FZA (contains an L195P substitution). The selection primer was the same for all mutagenesis reactions (5'-CGCGCTGTTAGCGGCGCCATTAAGTTCTGTCTCGGC-3') and changes a unique ApaI restriction site in the GST-RFXANK plasmid construct. All mutants were confirmed by DNA sequencing.

Immunoprecipitation and Western blotting. At >48 h after transfection, COS cells were harvested in 1 ml of lysis buffer (1% [vol/vol] NP-40, 10 mM Tris-HCl [pH 7.4], 150 mM NaCl, 2 mM EDTA, and 0.1% protease inhibitors) for 45 min at 4°C, and the amounts of the solubilized proteins were measured (BCA Protein Assay; Pierce, Rockford, Ill). Protein A-Sepharose (Amersham Pharmacia Biotech)-precleared lysates were subjected to immunoprecipitation using a rabbit polyclonal anti-Myc antibody (c-Myc [A-14]; Santa Cruz Biotechnology, Santa Cruz, Calif.). Immune complexes were recovered by binding to protein A-Sepharose beads during the overnight rotation at 4°C, resolved on a sodium dodecyl sulfate (SDS)-10% polyacrylamide gel, and transferred to a nitrocellulose membrane by a semidry technique. The membranes were immunostained with a mouse monoclonal anti-HA antibody (1:2,000; Boehringer Mannheim, Indianapolis, Ind.) followed by a horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G secondary antibody (1:2,000; Gibco-BRL, Rockville, Md.). Blots were developed by chemiluminescence assay (NEN Life Science Products, Boston, Mass.).

In vitro transcription and translation. The plasmids containing RFXAP (pT7T3-RFXAP), RFX5 (pcDNA3-RFX5), and CIITA (pSV-CIITA) cDNAs were transcribed and translated in vitro using the TnT T3-T7 coupled reticulocyte lysate system (Promega, Madison, Wis.) according to the manufacturer's instructions in the presence or absence of ³⁵S-labeled cysteine (NEN Life Science Products).

In vitro binding assays. GST fusion proteins were produced in Escherichia coli BL21(DE3)pLysS competent cells (Novagen, Madison, Wis.) during 4 h of induction with 1 mM IPTG (isopropyl--D-thiogalactopyranoside) and purified from total cell lysates with glutathione-Sepharose beads (Amersham-Pharmacia Biotech). For the GST pull-down assay, 10 µg of GST or GST fusion proteins was mixed with 10 µl of in vitro-translated proteins in 300 µl of binding buffer. The composition of the buffer for studying the interaction between RFXANK and CIITA was as follows: 50 mM Tris-HCl (pH 8.0), 5% glycerol, 0.5 mM EDTA, 5 mM MgCl₂, 1% bovine serum albumin, 500 mM NaCl, 0.25 Triton X-100, and 0.125% NP-40. When the interaction between RFXAP and RFXANK was studied, the detergent concentrations were increased to 1% Triton X-100 and 0.5% NP-40. After overnight incubation at 4°C, GST-coupled beads were washed five times with 1 ml of binding buffer. Bound proteins were eluted by boiling in SDS sample buffer. Proteins were resolved by SDS-10% polyacrylamide gel electrophoresis (SDS-10% PAGE) and revealed by autoradiography, and the signal was quantified as counts per minute.

EMSA. Electrophoretic mobility shift assays (EMSAs) were performed as described before (26).

Structure modeling. The structure of the ankyrin repeat domain of RFXANK (sequence number AAC69883) was modeled with the Swiss-Model approach for automated comparative protein modeling (28). As template files the ankyrin repeat-containing crystal structures of GABP (3) (RCBS accession code 1AWC; chain B; 2.15-Å resolution; Swiss-Prot database Q00421) and Swi6 (15) (1SW6; chain A; 2.10-Å resolution; P09959) were used. The sequence homology between the 125-amino-acid fragment of RFXANK (residues 119 to 243) and GABP (residues 33 to 157) corresponds to 28.8% identity (62.4% similarity). For RFXANK and Swi6 the sequence identity is about 26.8% (58.2% similarity) for the 67-amino-acid fragment of RFXANK (residues 88 to 154). For structure display and surface evaluation, hydrogen atoms were added to the model coordinates using the program X-PLOR (7). The fragments were assembled by a least-squares fit of the heavy-atom backbone coordinates of the overlapping residues 124 to 146.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

RFXANK has four ankyrin repeats. The 33-kDa RFXANK protein was the last recognized subunit of the RFX complex (22, 24). Besides its N-terminal PEST-like sequence and DNA-binding domain, it contains an ankyrin repeat domain at its C terminus. Three ankyrin repeats were reported to lie in this domain (22, 24), although one report suggested that there might be a fourth one, displaying weak homology to the general ankyrin repeat motif (11).

View larger version (44K): [in this window] [in a new window]   FIG. 1.   RFXANK contains four ankyrin repeats. (A) Sequence analysis of the ankyrin repeat domain of RFXANK. The secondary-structure elements (-hairpin loops, inner helix, turn, and outer helix) and the ankyrin repeat consensus sequence are displayed above the amino acid sequence of RFXANK residues 88 to 260. Identical and conserved residues relative to the ankyrin repeat consensus sequence are represented by white letters on a black background and by black letters on a dark gray background, respectively. A high degree of sequence similarity to the ankyrin consensus motif sequence can be observed from amino acid V117 to I242, suggesting the formation of four ankyrin repeats in RFXANK. (B) Schematic representation of RFXANK. RFXANK contains 260 amino acid residues and four ankyrin repeats at the C-terminal part of the protein.

Prediction of the three-dimensional structure of the ankyrin repeat domain of RFXANK. The first ankyrin repeat-containing protein with a determined three-dimensional structure was 53BP2, which interacts with the L-2 loop of the p53 tumor suppressor protein (17). As presented in a general model containing four ankyrin repeats (Fig. 2A, left panel), the ankyrin repeat domain consists of pairs of antiparallel (inner and outer) -helices that are stacked side by side and connected by a series of intervening -hairpin loops. The extended -sheet projects away from the helical pairs almost at right angles, resulting in a characteristic L-shaped cross-section (Fig. 2A, right panel). This assembled structure has been compared to a cupped hand: whereas the -hairpin loops form the fingers, the concave part, also termed the ankyrin groove, with solvent-exposed residues from the -helical bundles, forms the palm (30, 32). The structure is further stabilized by extensive intra- and interrepeat hydrogen bonds between the side chains.

View larger version (55K): [in this window] [in a new window]   FIG. 2.   Secondary-structure prediction of the ankyrin repeat domain of RFXANK. (A) Schematic representation of the secondary-structure elements of the ankyrin repeats in a three-dimensional view. Four ankyrin repeats represent only a general ankyrin domain structure. In the left scheme, -hairpin loops form the loop structures above the two planes of helices (inner and outer helices). The right scheme represents the same three-dimensional structure from a different perspective (as viewed from the first ankyrin repeat towards the last). The L-shaped structure appears, forming the ankyrin groove. -Hairpin loops, turns, and inner and outer helices form four different surfaces of the ankyrin repeat domain and are depicted with arrows. (B) Model structure of RFXANK residues 88 to 243. The ankyrin repeat domain of RFXANK is depicted as a ribbon structure, with two exposed variable residues at the very tip of each of the four -hairpin loops highlighted. This figure was generated with Molscript (19). N, amino terminus; C, carboxy terminus.

The -hairpin loops of the ankyrin repeats of RFXANK are required for binding to RFXAP. RFXANK and RFXAP, two subunits of the tripartite RFX complex, bind to each other strongly and specifically. We have shown previously that this interaction is the first step in the assembly of RFX (26). The C-terminal region of RFXAP, which contains a glutamine-rich domain, binds to RFXANK (11, 26). However, no mapping has been done on RFXANK.

View larger version (30K): [in this window] [in a new window]   FIG. 3.   -Hairpin loops of the ankyrin repeats of RFXANK are required for binding to RFXAP in vitro. The GST-RFXANK protein links 260 amino acids of RFXANK to GST and was used as a template for alanine mutagenesis. Four amino acid residues at the tips of the four -hairpin loops of RFXANK were mutated into alanines, and the resulting proteins were named GST-RFXANK-mut1 to4 (see Materials and Methods). In a GST pull-down assay, 35S-labeled RFXAP was incubated with GST alone or the wild-type and mutant GST-RFXANK fusion proteins and selected on glutathione-Sepharose beads. Bound proteins were separated by SDS-PAGE and revealed by autoradiography. RFXAP that was retained on the beads is depicted with an arrow. Lanes 1 to 6, results of the binding assay; lane 7, 25% of the input 35S-labeled RFXAP. Pluses above the autoradiographs indicate the presence of different proteins in the assay. GST alone (lane 1) and the wild-type (lane 2) and mutant (lanes 3 to 6) GST-RFXANK fusion proteins were equivalent and are presented in a Coomassie blue-stained SDS-polyacrylamide gel (GST input).

RFXANK binds simultaneously to RFXAP and CIITA in cells. In our preliminary studies we asked whether RFXANK interacts with proteins other than RFXAP. Under stringent conditions in vitro, we could not detect its binding to RFX5 (26). However, in vitro-transcribed and -translated CIITA was able to bind to bacterially produced hybrid GST-RFXANK protein in a GST pull-down assay (Fig. 4A, lane 2). The specificity of this binding was established because CIITA did not bind to GST alone (Fig. 4A, lane 1). In vitro studies for the binding of RFXAP and CIITA to RFXANK were done under more and less stringent binding conditions, respectively. In addition, only about 10% of input CIITA was retained by the hybrid GST-RFXANK protein (Fig. 4A, compare lanes 2 and 3). We conclude that although both can bind to RFXANK in vitro, RFXAP does so with higher affinity than CIITA.

View larger version (42K): [in this window] [in a new window]   FIG. 4.   RFXANK binds to CIITA both in vitro and in vivo. (A) RFXANK binds to CIITA in vitro. 35S-labeled CIITA was incubated with GST alone or the GST-RFXANK fusion protein and selected on glutathione-Sepharose beads. Retained CIITA is depicted with an arrow. Lanes 1 and 2, results of the binding assay; lane 3, 10% of the input 35S-labeled CIITA. Amounts of GST alone and GST-RFXANK fusion protein were the same as in Fig. 3 (lanes 1 and 2, GST input) and are therefore not presented. (B) RFXANK coprecipitates RFXAP and CIITA from the cells. The N terminus of RFXANK was linked to a Myc epitope tag, and the N termini of RFXAP and CIITA were linked to an HA epitope tag. Epitope-tagged proteins were expressed alone (lanes 1 to 3) or in different combinations (lanes 4 to 6) in COS cells. Precleared total cell lysates were immunoprecipitated (IP) with the anti-Myc antibody and protein A-Sepharose beads and examined for the presence of RFXAP and CIITA by Western blotting with the anti-HA antibody; 10% of precleared total cell lysates was analyzed for the presence of RFXANK, RFXAP, and CIITA (input). The same amount of total cell lysate from lane 6 was applied to lane 7 for easier identification of HA-tagged proteins (depicted with arrows). The asterisk depicts the unspecific band (lane 7).

Ankyrin repeats as structural modules are required for the binding of RFXANK to CIITA. To determine which part of RFXANK interacts with CIITA, we used the same approach as previously for studying its interaction with RFXAP. Ankyrin repeats were again the most likely candidate for the binding to CIITA. First, the mutant GST-RFXANK fusion proteins with substituted -hairpin residues were combined with in vitro- transcribed and -translated CIITA in a GST pull-down assay. As already shown in Fig. 4A, CIITA bound to the wild-type GST-RFXANK fusion protein but did not bind to GST alone (Fig. 5A, lanes 1 and 2). The binding persisted when all four mutant GST-RFXANK fusion proteins were used instead of the wild-type GST-RFXANK fusion protein. We conclude that CIITA does not interfere with the binding of RFXAP to the -hairpin loops of RFXANK.

View larger version (44K): [in this window] [in a new window]   FIG. 5.   The ankyrin repeat domain of RFXANK also binds to CIITA. (A) -Hairpin loops of RFXANK ankyrin repeats are not involved in binding to CIITA. The mutant GST-RFXANK-1 to 4 fusion proteins were used in a GST pull-down assay. 35S-labeled CIITA was incubated with GST alone or with the wild-type and mutant GST-RFXANK fusion proteins and selected on glutathione-Sepharose beads. Retained CIITA is depicted with an arrow. Lanes 1 to 6, results of the binding assay. Pluses above the autoradiographs indicate the presence of different proteins in the assay. Amounts of GST alone together with the wild-type and mutant GST-RFXANK fusion proteins were the same as in Fig. 3 (GST input) and are therefore not presented; 10% input 35S-labeled CIITA was the same as in Fig. 4A, lane 3. (B) Ankyrin repeats as structural units are required for CIITA binding. The first 251, 213, 180, and 147 amino acid residues of RFXANK represent the mutant RFXANK proteins that retain four, three, two, and one (the first) ankyrin repeat(s), respectively, and are fused to GST. All four C-terminal deletion mutants of GST-RFXANK were used in a GST pull-down assay similar to that described for panel A. Lanes 1 to 6, results of the binding assay. The amounts of all of the GST fusion proteins were the same and are shown in the Coomassie blue-stained gel at the bottom of the panel (GST input).

The inner helix of the third ankyrin repeat of RFXANK contacts RFXAP. So far we had determined the residues of the ankyrin repeat domain of RFXANK that bind to RFXAP and shown that the ankyrin repeats also bind to CIITA. To map precisely the residues of RFXANK that bind to CIITA, we performed another series of alanine mutageneses with the GST-RFXANK fusion protein as a template. Recently, a study was performed that included some mutant RFXANK proteins with point mutations in the ankyrin repeat domain (11). However, in that study the conserved structural residues of ankyrin repeats were mutated to alanines, causing a destruction of the ankyrin repeat domain. In contrast, we wanted to mutate variable residues of ankyrin repeats that should elucidate additional specific interactions between RFXANK and its binding partners in the context of the intact ankyrin repeat domain.

View larger version (90K): [in this window] [in a new window]   FIG. 6.   RFXAP binds to RFXANK at an additional contact point. (A) Mutagenesis scheme for exposed variable amino acid residues in the ankyrin repeat domain of RFXANK. The GST-RFXANK fusion protein was a template for another series of alanine mutagenesis reactions. Exposed amino acid residues (other than the ones from the -hairpin loops) were selected on the basis of the predicted three-dimensional structure of the ankyrin repeat domain of RFXANK. All mutated residues are underlined. The GST-RFXANK fusion proteins with mutations in the outer helices (OH) of the first, second, and third ankyrin repeats were named GST-RFXANK-OH1, -OH2, and -OH3, respectively. The fusion proteins with mutations in the turn regions of the second, third, and fourth repeats were named GST-RFXANK-turn2, -turn3, and -turn4, respectively. The inner helix (IH) of the third ankyrin repeat was mutated to yield mutant GST-RFXANK-IH3 fusion protein. The point mutations were introduced as single mutations (turns 2 to 4) or in clusters (OH1 to3 and IH3). (B) Cluster mutations in the inner helix of the third ankyrin repeat of RFXANK abolish its binding to RFXAP. Seven mutants depicted in panel A were used in a GST pull-down assay. They were mixed with 35S-labeled RFXAP, selected on glutathione-Sepharose beads, separated by SDS-PAGE, and revealed by autoradiography. Retained RFXAP is depicted with an arrow. Lanes 1 to 7, results of the binding assay; lane 8, 25% of the input 35S-labeled RFXAP. The mutant GST-RFXANK fusion proteins were equivalent and are presented in a Coomassie blue-stained SDS-polyacrylamide gel (GST input). (C) Display of the sites of mutational analysis on the surface of the modeled ankyrin repeat domain of RFXANK. Displayed in orange are mutated residues in four -hairpin loops (1 to 4) at the top of the model and residues in the inner helix of the third ankyrin repeat (inner helix 3) in the middle part of the model. L, Y, V, and R represent four mutated residues in inner helix 3, which is involved in binding to RFXAP. Displayed in blue are mutated turn residues (D, K, and K [bottom of the model]). This figure was generated with GRASP (27). N, amino terminus; C, carboxy terminus.

The point mutation in RFXANK from the FZA BLS patient abolishes its binding to RFXAP. The mutant GST-RFXANK-IH3 fusion protein that was not able to bind to RFXAP (Fig. 6B) was created by alanine mutagenesis of a cluster of residues in the inner helix of the third ankyrin repeat of RFXANK. Four residues were replaced with alanines, namely, Leu 195, Tyr 196, Val 198, and Arg 199 (see also Fig. 6C). All of these residues have protruding side chains and could be involved in contacting RFXAP. Interestingly, the recently described FZA patient from the complementation group B of BLS has a point mutation in RFXANK that changes leucine at position 195 into proline, resulting in the loss of expression of MHC II molecules on the surface of the patient's immune cells (25). Since the proline residue is a so-called helix breaker, we had to extend this structural change to the other residues in the inner helix of the third ankyrin repeat. We speculated that a single point mutation in the FZA patient was responsible for the loss of binding to RFXAP.

View larger version (31K): [in this window] [in a new window]   FIG. 7.   Intact inner helix 3 of RFXANK is critical for the assembly of the RFX complex. (A) The FZA patient from BLS complementation group B carries a mutation in the inner helix of the third ankyrin repeat of RFXANK. The leucine residue at position 195 in RFXANK was mutated into a proline residue, and the mutant protein was fused to GST to yield the mutant GST-RFXANK-FZA fusion protein. The mutant protein was used together with the wild-type and mutant GST-RFXANK-OH1 and -IH3 fusion proteins in a GST pull-down assay similar to that for Fig. 6B. Retained RFXAP is depicted with an arrow. Lanes 1 to 5, results of the binding assay; lane 6, 25% of the input 35S-labeled RFXAP. Pluses above the autoradiographs indicate the presence of different proteins in the assay. GST alone (lane 1) and the wild-type (lane 2) and mutant (lanes 3 to 5) GST-RFXANK fusion proteins were equivalent and are presented in a Coomassie blue-stained SDS-polyacrylamide gel (GST input). (B) The mutation in RFXANK from the FZA patient blocks the assembly of the RFX complex on DNA. Wild-type RFX5 and RFXAP proteins were transcribed and translated in vitro using the rabbit reticulocyte system. Wild-type and mutant GST-RFXANK fusion proteins were produced in bacteria, mixed in different combinations with the other two subunits of RFX, and incubated with the 32P-labeled oligonucleotide containing the S and X boxes of the DRA promoter. Inputs of GST proteins were equal and the same as presented in Fig. 7 (GST input).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, we defined four ankyrin repeats of RFXANK. Next, we modeled and studied its three-dimensional structure on the basis of other known ankyrin repeat-containing proteins. Exposed variable residues were replaced with alanines. In this way, we were able to determine the surfaces of ankyrin repeats that interact with its two binding partners, RFXAP and CIITA. These surfaces are composed of scattered residues rather than continuous amino acid stretches. RFXAP contacts two surfaces of RFXANK: -hairpin loops of the first three ankyrin repeats and one helix in the ankyrin groove. Contact points are limited and were clearly pinpointed. In contrast, CIITA binds RFXANK via multiple residues in the outer helices of the last three ankyrin repeats, which are located on the opposite side from the ankyrin groove of RFXANK. Alanine mutagenesis successfully positioned the binding partners of RFXANK into a complex protein network on MHC II promoters. Finally, we connected our binding studies to a disease. The FZA patient with BLS carries a single point mutation within RFXANK (25), resulting in an amino acid change within the inner helix of the third ankyrin repeat that is required for the binding to RFXAP. This mutation blocked the interaction between RFXANK and RFXAP in binding assays in vitro and the assembly of the RFX complex on DNA in EMSA. Thus, our mapping elucidates the background of yet another BLS mutation, which is responsible for the absence of MHC II determinants on the surface of B cells.

At the beginning of our mapping studies we were unable to detect an interaction between RFX5 and RFXANK in a stringent in vitro system (26), although these two subunits coimmunoprecipitated within the RFX complex from cells (data not shown). Thus, RFX5 requires a combinatorial surface of RFXANK and RFXAP to form a stable RFX complex. In contrast, direct interactions with RFXANK were obvious for RFXAP and CIITA. Therefore, we concentrated on the interaction between RFXANK and RFXAP for its essential role in the assembly of the RFX complex and on CIITA, which bound to a different surface of the ankyrin repeats. Although the ankyrin repeats of RFXANK were required for the binding to CIITA, no single or clustered point mutation abolished it. Moreover, CIITA is held on MHC II promoters by multiple interactions (33), suggesting that each one is relatively weak. Thus, an already weak interaction between CIITA and RFXANK is the sum of multiple contacts with its last three ankyrin repeats, a feature that makes their fine mapping an extremely difficult if not impossible task. Therefore, attempts to combine single and/or clustered point mutations to map this interaction precisely will most probably remain uninformative.

In this study, we combined direct binding assays with principles of structural biology that provided an advantage of looking at the protein as a module that can be changed without affecting its stability and conformation. Therefore, prediction of the three-dimensional structure of RFXANK represented a more reliable system for fine mapping of protein-protein interactions with its binding partners. The mutant protein from the FZA patient with BLS confirmed the importance of the conserved secondary-structure elements within the ankyrin repeats of RFXANK. Indeed, the leucine at position 195 does not play a structure-determining role by itself but is exposed on the surface of the inner helix 3 and is involved in the binding to RFXAP (Fig. 6B and C). However, the point mutation in the FZA patient that changes this residue to a proline destabilized the inner helix of the third ankyrin repeat and severely impaired the binding to RFXAP, which prevented normal nucleation of the RFX complex. Therefore, our mutagenesis distinguished between mutations that abolished the binding to the ankyrin repeat domain directly without affecting its overall secondary structure, as in the case of binding via -hairpin loops, or indirectly by influencing its secondary structure.

Our data show that RFXAP binds to two different surfaces of RFXANK (Fig. 8). These two surfaces are located on the same face of the ankyrin repeat domain and comprise the ankyrin groove that is shielded from the upper side by the cluster of four -hairpin loops. It is easy to speculate that RFXAP fits into this groove much like a key fits into a lock and is stabilized in this position by interactions with the -hairpin loops. In sharp contrast, CIITA does not bind to the -hairpin loops of RFXANK but requires its last three ankyrin repeats. Therefore, CIITA binds to the opposite surface of RFXANK, which is composed of outer helices and turns.

View larger version (40K): [in this window] [in a new window]   FIG. 8.   Binding surfaces for RFXAP and CIITA on the model structure of the ankyrin repeat domain of RFXANK (residues 88 to 243). The arrows depict the secondary-structure elements of the ankyrin repeat domain that are required for binding to RFXAP. The predicted surface that contacts CIITA is also shown. 1, 2, and 3, -hairpin loops of the first, second, and third ankyrin repeats, respectively; IH3, inner helix of the third ankyrin repeat; N, amino terminus; C, carboxy terminus; *, position of the L195P mutation in the FZA patient.

Although there are no specific secondary-structure elements within the ankyrin repeats that would be required for the binding of partner proteins, some common features exist. In the literature, there are many examples of other ankyrin repeat-binding proteins with the same binding pattern as that between RFXANK and RFXAP. For example, -hairpin loops are a very common interaction site of ankyrin repeats. Since the inner two residues of this highly exposed motif (DxxG) are variable (Fig. 1A), they generate the specificity required for the recognition of different binding partners. All -hairpin loops are involved in the interaction between the ankyrin-containing GABP and its DNA-binding partner protein GABP (3). On the other hand, only the fourth -hairpin loop is involved in the interaction between the ankyrin-containing 53BP2 and its binding partner p53 (17). Another group of nonconserved residues are those lying on the exposed face of -helices in the ankyrin groove. Interactions between GABP and GABP as well as Cdk kinase activity inhibitors p16INK4a and p19INK4d that bind to Cdk6 are examples of this type of interaction (6, 29).

In addition, there are many ankyrin repeat-containing proteins with multiple binding partners. For example, the dimeric transcription factor NF-B interacts with its inhibitor I-B, which contains six ankyrin repeats (18). Two different domains of p65 as well as p50 bind to the ankyrin groove and -hairpin loops of I-B, respectively. Similarly, outer helices of ankyrin repeats can mediate protein-protein interactions (21). Therefore, all of the surfaces of ankyrin repeats of RFXANK that contact its binding partners have been verified in other systems. The architecture of the DNA-bound complex between RFX and CIITA and the central role of RFXANK in its assembly are summarized in our model in Fig. 9.

View larger version (71K): [in this window] [in a new window]   FIG. 9.   A model for RFXANK-mediated protein complex assembly on the X box of MHC II promoters. The -hairpin loops of the first three ankyrin repeats of RFXANK interact with RFXAP, while the outer helices of the last three ankyrin repeats on the opposite face of the ankyrin repeat domain contact CIITA. The interaction between RFXAP and the inner helix of the third ankyrin repeat of RFXANK is not shown for simplicity of the model. Ankyrin repeats of RFXANK are depicted with numbers 1 to 4. N, amino terminus; C, carboxy terminus.

BLS is a unique genetic disease with a highly heterogeneous genetic background resulting in severe combined immunodeficiency. In general, different BLS mutations result in a disease that is more or less severe, depending on the amount of residual MHC II molecules on the surface of patient's B cells. This polymorphism could result from residual binding and activity of mutated proteins. This possibility was confirmed by our in vitro binding assays and EMSA with the mutant RFXANK protein from the FZA patient (Fig. 7). Moreover, the overexpression of CIITA can increase the surface expression of MHC II molecules on gamma interferon-treated FZA fibroblasts (25). On the other hand, large deletions of proteins that are common in most BLS patients cannot be compensated for unless mutated proteins are replaced by their wild-type counterparts. Finally, BLS has taught us a lot about the assembly of regulatory complexes on MHC II promoters and eukaryotic transcription. Although mutations in ankyrin repeats had been connected to a disease, namely, cancer (17, 30), they arose in somatic cells. To our knowledge, BLS is the first congenital disease that targets the ankyrin repeats.